The present invention generally relates to solid surfaces that are usefully exposed to flowing solute, and particularly to articles such as capillaries or microchips that have a polymer adsorbed to their surfaces which is effective to reduce electroosmotic flow when the articles are used in electrophoretic separations.
Electrophoresis is a well-known technique for the separation of charged species by utilizing their differences in rate of migration under the influence of an electrical field. The advantages associated with capillary electrophoresis are numerous. Quantitative information can be achieved with very small sample sizes, and the amount of gel or buffer consumed is minuscule. Capillary electrophoresis is associated with certain phenomenon which are not present in traditional slab gel electrophoresis. One of these is the now familiar electroosmotic flow phenomenon characterized by bulk flow of buffer solutions toward one of the electrodes.
For many electrophoretic applications, electroosmotic flow is undesirable and eliminating or substantially reducing the bulk flow is preferred. Generally, when electroosmotic flow is reduced to a minimum, electrophoretic sample components move only by electrophoretic migration, which improves analysis reproducibility and mass recovery of sample components.
Jorgenson and Lukacs had noted that separation of model proteins, such as cytochrome, lysozyme, and ribonuclease A, in untreated fused silica capillaries with a phosphate buffer at pH 7 was accompanied by strong tailing, and suggested this might be caused by Coulombic interactions of the positively charged proteins and the negatively charged capillary wall. (Jorgenson et al., Science, 222, 1983, pp. 266-272.) The authors reported investigating Teflon capillaries, but found these also exhibit significant adsorptivity toward proteins. They attempted to deactivate the surface of fused silica with groups such as trimethyl silane, octadecylsilane, aminopropylsilane, and cross-linked methyl cellulose, which apparently did not work. They then turned to bonding glycol-containing groups to the surface.
Lauer and McManigill, Analytical Chemistry, 58, 1986, p. 166, reported that the Coulombic repulsion between proteins and the capillary wall of silica capillaries can overcome adsorption tendencies of the proteins with the capillary wall. They demonstrated separations of model proteins (ranging in molecular weight from 13,000 to 77,000) by varying the solution pH relative to the isoelectric point (pI) of the proteins to change their net charge. However, disadvantages of this approach are that silica begins to dissolve above pH 7, which shortens column life and degrades performance, and only proteins with pI""s less than the buffer pH can be analyzed.
Yet another approach to the problem of undesirable protein interactions with the capillary wall is described by U.S. Pat. No. 4,680,201, inventor Hjerten, issued Jul. 14, 1987, wherein a method for preparing a thin-wall, narrow-bore capillary tube for electrophoretic separations is provided by use of a bifunctional compound in which one group reacts specifically with the glass wall and other with a monomer taking part in a polymerization process. This free-radical procedure results in a polymer coating, such as polyacrylamide coating, and is suggested for use in coating other polymers, such as poly(vinylalcohol) and poly(vinylpyrrolidone).
Other covalently bound species have subsequently been described. U.S. Pat. No. 5,605,613, issued Feb. 25, 1997, inventor Shieh, discloses capillary columns having a neutral cross-linked hydrophilic coating on the interior wall surfaces, which is said to reduce analyte interaction with the interior surface. The coated column includes a polymer covalently bound to the interior surface.
U.S. Pat. No. 5,840,388, issued Nov. 24, 1998, inventors Karger et al., describes a coated microcapillary column for high performance electrophoresis in which a polymeric coating layer is formed by polymerizing an organic compound such as polyvinyl alcohol to the column surface. U.S. Pat. No. 5,792,331, issued Aug. 11, 1998, inventors Srinivasan et al., discloses a method of coating a capillary or chromatography packing by covalently bonding a polymer such as poly(vinylpyrrolidone) (xe2x80x9cPVPxe2x80x9d) to capillary walls.
Although capillary treatments involving chemical bonding (that is, covalent bonding) can function to reduce electroosmotic flow, the treatment processes are relatively time consuming and expensive, and also tend to create relatively thick coatings on the interiors of the capillary columns. Capillary columns used in capillary electrophoresis typically are fabricated of lengths of silica tubing having an inner diameter on the order of 25 xcexcm to 200 xcexcm, and thus the covalently bonded coatings can significantly increase the time for achieving electrophoretic separations.
U.S. Pat. No. 5,552,028, issued Sep. 3, 1996, inventors Madabhushi et al., discloses a composition for separating polynucleotides in which one component of the separation medium includes a silica-adsorbing polymer; and, U.S. Pat. No. 5,567,292, issued Oct. 22, 1996, inventors Madabhushi et al., discloses a method of suppressing electroosmotic flow by which a separation medium is provided that contains a silica-adsorbing polymer in a concentration of the separation medium in a range between about 0.001% and about 10% wt./v. These two Madabhushi et al. patents thus disclose a type of dynamic coating methods, whereby the eluent, or separation medium, itself contains additives for coating during the separations so as to mask surface charges; however, these additives may interact with the analytes which can lead to some unexpected and undesired results, and optimization tends to be limited to the use of certain specific separation matrices.
It is an object of the present invention to provide solid surfaces, such as capillary tubes that are useful for electrophoretic separations, where interactions between solutes flowed along the surfaces are considerably reduced, while preparation of the inventive surfaces is simple, fast, relatively inexpensive yet results in long-term stability.
Further objects and advantages of the invention will become apparent to those skilled in the art upon examination of the specification and appended claims, as well as in practice of the present invention.
In one aspect of the present invention, an article of manufacture is provided that is useful in differentiating between solutes, such as when the article is exposed to a flow of solutes during electrophoretic separations where the solutes include charged species such as proteins and oligonucleotides. Particularly preferred articles of the invention are formed as capillary tubes and are useful in DNA sequencing analysis, DNA fragment analysis and sizing, and protein separation and analysis. The inventive articles have a solid surface that carries a polymer. The polymer is adsorbed to the surface and functions to reduce interactions with the surface. The adsorbed polymer preferably is a polylactam, most preferably is poly(vinylpyrrolidone), or PVP, and preferably with a molecular weight of greater than about 1,000,000 daltons (weight-average) which has been simply and quickly coated by adsorption onto the inner wall of capillaries prior to introduction of the separation medium.
Surfaces treated in accordance with the invention have reduced electroosmotic flow, and may be used in virtually any capillary electrophoretic separation, where it is desirable to minimize or eliminate electroosmotic flow. The inventive surfaces are particularly useful as coated capillary columns in electrophoretic separation systems such as the CEQ2000, P/ACE MDQ, and Paragon CZE 1000 systems manufactured and sold by Beckman Coulter, Inc., Fullerton, Calif. for applications such as in DNA sequencing analysis, DNA fragment analysis and sizing, and protein separation and analysis.